Hereditary hemochromatosis: the clinical significance of the S65C mutation

Hereditary hemochromatosis (HH) is a common genetic disease with iron overload in certain organs, especially the liver. Most cases are homozygous for the C282Y mutation in the HFE gene; a few are C282Y heterozygous, compound C282Y/H63D heterozygous, or have no known mutation. A third mutation, S65C, has been associated with HH, but this finding is disputed. We have studied the clinical significance of various genotypes with the S65C mutation. In a population-based screening for HH in 65,238 persons, 613 had high serum transferrin saturation in two blood samples and were invited for HFE genotyping. In 556 persons with complete data sets, we studied the serum ferritin concentration and the risk of being diagnosed with phenotypic HH in the various genotypic groups. The phenotypic diagnosis was given without knowing the genotypic result. Except for the C282Y homozygotes, no differences in median serum ferritin concentrations were found between the various genotypic groups. However, the C282Y/S65C compound heterozygous group had a higher risk of being diagnosed with phenotypic HH than the wild-type group, as did the C282Y homozygous and the C282Y/H63D compound heterozygous groups. When combined with the C282Y mutation, the S65C mutation is associated with an increased risk of being diagnosed with phenotypic HH.     

Interactions of the ectodomain of HFE with the transferrin receptor are critical for iron homeostasis in cells

Expression of wild type HFE reduces the ferritin levels of cells in culture. In this report we demonstrate that the predominant hereditary hemochromatosis mutation, C282Y(2) HFE, does not reduce ferritin expression. However, the second mutation, H63D HFE, reduces ferritin expression to a level indistinguishable from cells expressing wild type HFE. Further, two HFE cytoplasmic domain mutations engineered to disrupt potential signal transduction, S335M and Y342C, were functionally indistinguishable from wild type HFE in this assay, as was soluble HFE. These results implicate a role for the interaction of HFE with the transferrin receptor in lowering cellular ferritin levels.     

Prevalence of the C282Y, H63D, and S65C mutations of the HFE gene in 1,146 newborns from a region of Northern Spain

In Spain, 85% of patients with genetic hemochromatosis (GH) are homozygous for the C282Y mutation of the HFE gene. H63D and S65C mutations of HFE may also play some role in the disease. The aim of this study was to establish the prevalence of C282Y, H63D, and S65C mutations of the HFE gene in newborns in Catalonia, Spain. One thousand one hundred forty-six newborn screening cards were selected randomly. DNA from these cards was extracted and HFE mutations were analyzed with the LightCycler equipment (Roche Diagnostics Gmbh, Mannheim, Germany). Sufficient DNA sample was obtained to screen for the three mutations in 1,043 cases (91%). The allelic frequencies of C282Y, H63D, and S65C mutations were 0.03 (IC 95% 0.022-0.037), 0.2 (IC 95% 0.19-0.22), and 0.01 (95% confidence interval [CI] 0.006-0.015), respectively. The frequency of C282Y homozygous newborns was 0.001 (95% CI 0.0005-0.0014). The frequencies of newborns doubly heterozygous for C282Y/H63D and C282Y/S65C were 0.01 (95% CI 0.005-0.02) and 0.002 (95% CI 0.0002-0.01), respectively. The allelic frequency of C282Y mutation is similar to that observed in Southern France, in the Czech Republic and in some areas of Italy. The allelic frequency of H63D mutation in Catalonia is the highest reported to date. Nevertheless, S65C is infrequent. These data should be kept in mind when designing hemochromatosis genotypic screening programs in Catalonia.     

Effects of HFE C282Y and H63D polymorphisms and polygenic background on iron stores in a large community sample of twins

The aim of this study was to assess and to compare the role of HFE polymorphisms and other genetic factors in variation in iron stores. Blood samples were obtained from 3,375 adult male and female twins (age range 29-82 years) recruited from the Australian Twin Registry. There were 1,233 complete pairs (562 monozygotic and 571 dizygotic twins). Serum iron, transferrin, transferrin saturation with iron, and ferritin were measured, and the HFE C282Y and H63D genotypes were determined. The frequency of the C282Y allele was.072, and that of the H63D allele was.141. Significant sources of variation in the indices of iron status included age, sex, age-sex interaction, body-mass index, and both the C282Y and H63D genotypes. The iron, transferrin, and saturation values of CC and CY subjects differed significantly, but the ferritin values did not. After correction for age and body-mass index, 23% and 31% of the variance in iron, 66% and 49% of the variance in transferrin, 33% and 47% of the variance in transferrin saturation, and 47% and 47% of the variance in ferritin could be explained by additive genetic factors, for men and women, respectively. HFE C282Y and H63D variation accounted for <5% of the corrected phenotypic variance, except for saturation (12% in women and 5% in men). We conclude that HFE CY and HD heterozygotes differ in iron status from the CC and HH homozygotes and that serum transferrin saturation is more affected than is serum ferritin. There are highly significant effects of other as-yet-unidentified genes on iron stores, in addition to HFE genotype.     

MutantHFE genotype leads to significant iron overload in patients with liver diseases from western Romania

The present study aimed at assessing the frequency ofHFE mutations (C282Y, H63D and S65C) in western Romanian patients with liver disease of diverse aetiologies suspected of iron overload. A total of 21 patients, all Romanian residents hospitalized with clinical suspicion of iron overload and liver disease, were assayed for C282Y, H63D and S65C mutations, serum ferritin and viral hepatitis markers. Overall, 9 out of the 21 patients (42.86%) were found to harbour mutations in theHFE gene: 4 homozygotes C282Y (19.0%), 1 compound heterozygote C282Y/H63D (4.8%), 1 single heterozygote C282Y (4.8%), 2 single heterozygotes H63D (9.5%), 1 single heterozygote S65C (4.8%), and 12 wild-type cases (57.1%). Among the subgroup of 10 patients with the most prominent signs of iron overload (hyperferritinaemia and/or hepatocyte iron score ≥ 1), without hepatocellular carcinoma, theHFE genotypes were conclusive in 5 cases (50%). They had significantly increased ferritin levels compared to wild-type cases (P = 0.029). The inclusion of iron studies during routine clinical visits, coupled with the availability ofHFE genotyping for family and population studies, should facilitate the early detection of hereditary haemochromatosis in Romania.     

Hemochromatosis (HFE) and transferrin receptor-1 (TFRC1) genes in sporadic porphyria cutanea tarda (sPCT).

Porphyria cutanea tarda (PCT), a disorder characterized by a photosensitive dermatosis and hepatic siderosis, is caused by a decreased activity of the hepatic enzyme uroporphyrinogen decarboxylase (UROD). Two forms of PCT have been described: a familial one (fPCT) with an inherited decrease of UROD activity in all tissues and a sporadic one (sPCT) with a decreased UROD activity restricted to the liver. Iron overload and acquired factors including hepatic viral infections, alcohol, drugs contribute to the expression of PCT. In 65 French sPCT patients and 108 controls we have evaluated the respective role of iron and HCV status, the hemochromatosis (HFE) gene mutations frequencies (H63D. S65C, C282Y), and in a case control study we searched for an association between sPCT and the human transferrin receptor-1 (TFRC1) gene whose product is thought to be in functional association with the HFE protein: three single nucleotide polymorphisms (SNPs) previously characterized and 2 novel ones were studied. The iron-related parameters and transaminases were higher in sPCT patients than those of non-porphyric controls. Of the sPCT patients studied, 28% were HCV positive. In the HFE gene, 17% of sPCT patients carried C282Y mutation compared to 4% in controls, no significant differences were found with H63D and S65C variants. Compound heterozygous genotypes, C282Y/H63D or C282Y/S65C, were not significantly different in sPCT and control groups. Independently from HFE gene mutations, an association was found between the IVS4+198 T allele in the TFRC1 gene and sPCT patients. Analysis of HFE genotypes indicated that C282Y (but not H63D nor S65C) is a susceptibility factor for the development of sPCT in West European continental patients. However, analysis of TFRC1 genotypes suggest that sPCT should be considered as a multifactorial disorder in which other intracellular iron metabolism genes could be involved.     

Initial screening transferrin saturation values, serum ferritin concentrations, and HFE genotypes in whites and blacks in the Hemochromatosis and Iron Overload Screening Study

We compared initial screening data of 44,082 white and 27,124 black Hemochromatosis and Iron Overload Screening (HEIRS) Study participants. Each underwent serum transferrin saturation (TfSat) and ferritin (SF) measurements without regard to fasting, and HFE C282Y and H63D genotyping. Elevated measurements were defined as: TfSat more than 50% (men), more than 45% (women); and SF more than 300 ng/ml (men), more than 200 ng/ml (women). Mean TfSat and percentages of participants with elevated TfSat were significantly greater in whites than in blacks. Mean SF and percentages of participants with elevated SF were significantly greater in blacks than in whites. TfSat and SF varied by gender and age in whites and blacks. Prevalences of genotypes that included either C282Y or H63D were significantly greater in whites than in blacks. The prevalence of elevated TfSat and SF plus genotypes C282Y/C282Y, C282Y/H63D, or H63D/H63D was 0.006 in whites and 0.0003 in blacks. Among whites with HFE C282Y homozygosity, 76.8% of men and 46.9% of women had elevated TfSat and SF values. Three black participants had HFE C282Y homozygosity; one had elevated TfSat and SF values. Possible explanations for differences in TfSat and SF in whites and blacks and pertinence to the detection of hemochromatosis, iron overload, and other disorders with similar phenotypes are discussed.     

Genetic hemochromatosis, a Celtic disease: is it now time for population screening?

In populations of northern European ancestry, hereditary hemochromatosis (HH) is tightly linked to mutations within the hemochromatosis gene (HFE gene). Over 93% of Irish HH patients are homozygous for the HFE gene C282Y mutation, providing a reliable diagnostic marker of the disease in this population. However, the prevalence of the C282Y mutation and that of the second HFE gene mutation, H63D, have yet to be determined within the Irish population. The objective of this study was to identify the true prevalence of the genetic form of HH in the Irish population. DNA was extracted from 1002 randomly selected newborn screening cards and analyzed for the C282Y and H63D mutations within the HFE gene. Complete results were obtained from 800 cards. Mutations were identified in 364 (46%) neonates. Eight (1%) neonates were homozygous for C282Y and 8 (1%) were homozygous for H63D. One hundred and fifty-five (19%) neonates were C282Y heterozygous and 226 (28%) were H63D heterozygous. Of these, 33 (4%) carried one copy of both C282Y and H63D mutations, i.e., compound heterozygous. Allele frequencies for C282Y and H63D were 11% and 15%, respectively. The high C282Y allele frequency in the Irish population together with its close linkage to HH indicate that C282Y genotyping is the preferred screening strategy for this disease in Ireland.     

Frequency of HFE gene mutations C282Y and H63D in Bosnia and Herzegovina

Genetic epidemiology studies of hereditary hemochromatosis (HHC) have shown a high prevalence of the C282Y mutation in individuals of the North Western European origin, whereas lower prevalence of HFE gene mutations was detected in the populations from southern European countries. However, no HFE mutation prevalence data have been provided for the population of Bosnia-Herzegovina so far. Therefore, the aim of this study was to determine the frequency of the C282Y and H63D HFE gene mutations in the population of Bosnia-Herzegovina. Among 200 analysed subjects 8 (4%) were C282Y heterozygotes; no C282Y homozygotes were found. The frequency of the H63D allele was 11.5%. There were 33 (16.5%) heterozygotes and 6 (3%) homozygotes for the H63D mutation. One (0.5%) compound heterozygote C282Y/H63D was identified. The observed C282Y and H63D allele frequency was 2.25% (95% confidence interval: 1.2-4.2) and 11.5% (95% confidence interval: 8.7-14.9), respectively. The prevalence of the C282Y and H63D mutations was estimated in Bosnia-Herzegovina, which fit well in the European northwest-to-southeast gradient of the C282Y mutation distribution. In addition, these results have an important implication for clinical evaluation of HHC in Bosnia-Herzegovina.     

Unique frequencies of HFE gene variants in Roma/Gypsies

The aim of this study was to assess the frequencies of three hemochromatosis gene (HFE) mutations in ethnic Roma/Gypsies in Slovakia. A cohort of 367 individuals representing general population and not preselected for health status was genotyped by TaqMan real-time PCR assay for C282Y, H63D and S65C mutations in HFE gene. A unique genetic profile was revealed: C282Y is found in the highest frequency of all Central European countries (4.90%), while the frequency of H63D mutation (4.09%) is lower than any reported in Europe so far. S65C mutation was not present in the cohort. These mutation frequencies can be explained rather by gene influx and genetic isolation than by genetic inheritance from a former Roma/Gypsy homeland.     

Frequency of HFE H63D, S65C, and C282Y mutations in patients with iron overload and controls from Toledo, Spain

Hereditary hemochromatosis (HH) is an autosomal recessive disease caused by a defective iron absorption. C282Y is the most frequent HFE gene mutation causing HH in Northern European populations and their descendants. However, two other mutations, H63D and S65C, have been described as pathogenic changes. In this study, we have tried to evaluate the frequency of these three mutations in our community. Eighty-three patients with clinical and/or biochemical features of hemochromatosis and 150 controls were screened for H63D, S65C, and C282Y mutations using a PCR-restriction fragment length polymorphism (RFLP)-based strategy. In contrast to previous studies, 7% of the patients were homozygous for C282Y mutation. The remaining patients were 20% H63D homozygous, 10% H63D/C282Y compound heterozygous, 1% H63D/S65C compound heterozygous, 22% H63D heterozygous, 2% C282Y heterozygous, 2% S65C heterozygous, and 36% of patients lacked any of the three mutations studied, despite the fact that they showed clinical/biochemical features of hemochromatosis. We observed a high frequency of the H63D mutation in both the control group and patients, whereas the main genotypes implicated in HH in our series were H63D homozygous and H63D/C282Y compound heterozygous. We propose that the H63D mutation be analyzed in HH patients from our geographic area. Moreover, further studies are needed to elucidate the role of this mutation in the development of HH and the genetic, environmental or other factors that affect the genotype-phenotype correlation between H63D and hemochromatosis.     

The Q283P amino-acid change in HFE leads to structural and functional consequences similar to those described for the mutated 282Y HFE protein

In Caucasians, 4–35% of hemochromatosis patients carry at least one chromosome without a common HFE mutation (i.e. C282Y, H63D and S65C). Several studies have now shown that iron overload phenotypes in such patients can be associated with uncommon HFE mutations. We previously supported implication of the C282Y/Q283P compound heterozygous genotype in hemochromatosis phenotypes and, based on molecular dynamics simulations, proposed that the Q283P substitution prevents normal folding of the HFE ₃-domain. In the current work, we have used HeLa cells carrying wild-type or Q283P-mutant HFE cDNA under the control of a tetracycline-sensitive promoter to functionally characterise the Q283P mutation. Experiments using cells over-expressing wild-type HFE confirm the existence of ₂microglobulin(₂m)/HFE and HFE/transferrin receptor 1 (TfR1) interactions, as well as the capacity of HFE to reduce transferrin-mediated iron uptake. In contrast, neither ₂m/HFE nor HFE/TfR1 complex formation was detected in cells over-expressing the mutated form of HFE. Moreover, the 283P HFE protein was found to have a very limited effect on the major cellular iron uptake pathway. Combined, our results indicate that the Q283P mutation leads to structural and functional consequences similar to those described for the main hereditary hemochromatosis mutation. As a consequence, our study has implications for the screening of hemochromatosis patients that have one or two copies of HFE which lack the main mutations. It also highlights that protein structure prediction methods could be more generally used to better interpret relationships between rare genotypes and molecular diagnosis of a human inherited disorder.     

HFE genotype frequencies in consecutive reference laboratory specimens: comparisons among referral sources and association with initial diagnosis

We quantified HFE genotype frequencies in specimens submitted by physicians grouped by specialty and determined associations of genotypes with initial diagnosis based on phenotyping in patients evaluated at an iron disorders center. Of 526 specimens (519 from Alabama), these "typical" hemochromatosis-associated genotypes were detected: 85 C282Y/C282Y, 50 C282Y/H63D, and 27 H63D/H63D. Respective frequencies of C282Y/C282Y in specimens from an iron disorders center (n = 156), gastroenterologists (n = 147), hematologists/medical oncologists (n = 85), liver transplant surgeons (n = 11), endocrinologists and rheumatologists (n = 9), and "other sources" (n = 7) were greater (p < 0.05) than in population controls. In 44 patients from an iron disorders center initially diagnosed as "presumed hemochromatosis," 27 (61.4%) had C282Y/C282Y, 10 (22.7%) had C282Y/H63D, and 3 (6.8%) had H63D/H63D. C282Y/C282Y was not detected in 48 patients with "abnormality probably not an iron overload disorder." A total of 20.5% of 44 family members of patients had "typical" hemochromatosis-associated HFE genotypes (7.0% controls; p = 0.02). We conclude that most physicians who submitted specimens identify patients by phenotyping who have greater frequencies of "typical" hemochromatosis-associated HFE genotypes than controls, and that HFE mutation testing is useful in detecting hemochromatosis in family members of persons with hemochromatosis or iron overload.     

A primer for predicting risk of disease in HFE-linked hemochromatosis

Since the discovery of the hemochromatosis gene (HFE) in 1996, there has been increasing interest in diagnostic testing for the C282Y and H63D mutations. The high frequency of these two alleles and their incomplete penetrance in homozygotes and compound heterozygotes make genetic counseling for hemochromatosis different from some other autosomal recessive conditions in that parents and children may also be at risk for iron overload, while homozygotes may remain asymptomatic. We provide a guideline for genetic counseling in HFE-linked hemochromatosis based on the genetic probability of inheriting HFE mutations and known information about expression of iron overload in various HFE genotypes. Genetic probabilities were based on allele frequencies derived from large population studies and Hardy-Weinberg equilibrium estimates. Expression of iron overload in those of various genotypes was based on available estimates of serum ferritin from population screening studies. Estimates for the likelihood of clinical iron overload requiring follow-up screening or treatment are provided by gender and genotype. The probability of inheriting HFE mutations and developing iron overload can be estimated in family members of a proband with HFE mutations. Many C282Y homozygotes will not have clinical iron overload. The risk is highest in men and their C282Y homozygous brothers and significantly lower in homozygous women. Iron overload is uncommon in compound heterozygotes and H63D homozygotes.     

Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells

The hereditaryhemochromatosis protein HFE is known to complex with the transferrinreceptor; however, its function regarding endocytosis of transferrin isunclear. We performed patch-clamp capacitance measurements intransfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNAunder the control of a tetracycline-sensitive promoter. Whole cellexperiments in cells with suppressed expression of wild-type HFErevealed a decrease in membrane capacitance, reflecting predominance ofendocytosis in the presence of transferrin. Cells overexpressingC282Y-mutant HFE displayed less intense capacitance decreases, whereasno significant decrease was observed in cells overexpressing wild-typeHFE. The formation of single endocytic vesicles in cells withsuppressed expression of wild-type HFE was greatly increased in thepresence of transferrin as revealed by cell-attached recordings.According to their calculated diameters, many of these vesiclescorresponded to clathrin-coated vesicles. These results suggest thatwild-type HFE negatively modulates the endocytic uptake of transferrin.This inhibitory effect is attenuated in cells expressing C282Y-mutantHFE. Time-resolved measurements of cell membrane capacitance provide apowerful tool to study transferrin-induced endocytosis in single cells.

     

Prevalence of C282Y and H63D mutations in the haemochromatosis (HFE) gene in Tunisian population

The studies of the HFE mutations: H63D and C282Y in North African populations have revealed the extreme rarity or even the absence of the C282Y mutation. We have examined 1140 chromosomes (570 Tunisian people) for the presence of the two HFE mutations by PCR-RFLP analysis. We have found that the allele frequencies are, respectively, 15.17% (+/-2.1%) for the H63D and 0.09% (+/-0.17%) for the C282Y. These results are consistent with the worldwide spread of the H63D mutation and the north European restriction of the C282Y. This study will be completed by determining whether homozygote trait for H63D and associated risk factors (beta thalassémia) can lead to iron overload in Tunisia.     

Protective role of calreticulin in HFE hemochromatosis

HFE gene mutations are associated with over 80% of cases of hereditary hemochromatosis (HH), an iron-overload disease in which the liver is the most frequently affected organ. Research on HFE has traditionally focused on its interaction with the transferrin receptor. More recent studies have suggested a more complex function for this nonclassical MHC-I protein. The aim of this study was to examine how HFE and its two most common mutations affect the expression of selected genes in a hepatocyte-like cell line. Gene expression was analyzed in HepG2 cells overexpressing wild-type and mutant HFE. The effect of HFE in iron import and oxidative stress levels was assessed. Unfolded protein response (UPR)-activated gene expression was analyzed in peripheral blood mononuclear cells from characterized HH patients. C282Y HFE down-regulated hepcidin and enhanced calreticulin mRNA expression. Calreticulin levels correlated with intracellular iron increase and were associated with protection from oxidative stress. In C282Y(+/+) patients calreticulin levels correlated with the expression of the UPR marker BiP and showed a negative association with the number of hereditary hemochromatosis clinical manifestations. The data show that expression of C282Y HFE triggers a stress-protective response in HepG2 cells and suggest a role for calreticulin as a modifier of the clinical expression of HH.     

Hereditary hemochromatosis in Spain

The C282Y mutation of the HFE gene has been reported as the main cause of hereditary hemochromatosis (HH). Another missense mutation (H63D) has also been detected at an increased frequency in a compound heterozygote state with the C282Y mutation in HH patients. However, these two mutations are not present in all of the HH patients, indicating that other mutations in the HFE gene, or in other loci, should exist. The present study reports the frequencies of the C282Y and H63D mutations in 74 Spanish HH patients and the results of the sequencing analysis of the HFE exons, intron-exon boundaries, and 588 bp of the 5' region in 5 patients negative for the C282Y mutation. We have detected a high frequency of the C282Y mutation (85.1%) in Spanish HH patients, indicating that this mutation is the most common defect associated with the disease in Spain. The screening of the HFE regions in our patients without the C282Y mutation has revealed the presence of five polymorphisms. However, no other pathological mutations have been found. Therefore, further efforts to characterize the unscreened part of the HFE gene or other loci should be taken to identify the potential genetic factors causing HH in the C282Y-negative patients.     

Comparison of oxidative stress and the frequency of polymorphisms in the HFE gene between hemoglobin S trait blood donors and sickle cell disease patients

It is well documented that Hb S and iron affect blood cells, and trigger oxidative processes and generation of free radicals with potential for lipid peroxidation. We evaluated the frequency of polymorphisms in the HFE gene in Hb AS blood donors and how these polymorphisms influenced lipid peroxidation and antioxidant capacity. Blood samples were collected from 211 Hb AS blood donors, 119 Hb AA blood donors as a control group, and 28 sickle cell disease patients (Hb SS). The H63D allele was found at a frequency of 10.5% in the Hb AS samples, and the C282Y allele frequency was 0.7%. In the control group, the frequencies of the H63D and C282Y alleles were 13.4 and 2.1%, respectively. In the sickle-cell disease patients, the H63D and the C282Y allele frequencies were 10.7 and 3.5%, respectively. The frequencies of the C282Y and H63D polymorphisms in Hb AS blood donors are similar to those reported for the Brazilian population. Serum malondialdehyde values, indicative of lipid peroxidation, were highest in sickle cell patients, independent of the polymorphisms in the HFE gene, with significant differences, showing the influence of Hb S allele in the levels of lipid peroxidation. However, the trolox equivalent antioxidant capacity average levels, indicative of the antioxidant capacity, were reduced with significant differences, indicating that in spite of a lipid peroxidation raise, this is not followed by the increased of the antioxidant capacity, leading to oxidative stress.